Method for dna or rna amplification from urine, saliva and/or mouthwash samples

Abstract

A method for sample preparation and/or DNA amplification from urine, saliva and/or mouthwash samples is disclosed. The method involves providing a sample preparation system comprising a filter and providing a urine, saliva and/or mouthwash sample, then filtering at least a portion of the sample through the filter, thereby retaining bacteria, fungi, viruses protists and/or cancerous or precancerous cells in the filter. Subsequently a lysis reagent is applied to the filter. Finally, the DNA found in the lysate is amplified.

1 . A method for sample preparation and DNA or RNA amplification from a urine sample, a saliva sample and/or a mouthwash sample by a user, wherein the method comprises the steps of
 (i) providing a sample preparation system comprising a filter,   (ii) providing a urine sample, a saliva sample and/or a mouthwash sample,   (iii) filtering at least a portion of the sample through the filter by applying a pressure on the sample that is above atmospheric pressure, thereby retaining bacteria, fungi, viruses, protists and/or cancerous or precancerous cells in the filter,   (iv) applying a lysis reagent to the filter,   (v) amplifying DNA or RNA of the bacteria, fungi, viruses, protists and/or cancerous or precancerous cells contained in the lysate,   wherein the user performs at least steps (ii) to (v).   
     
     
         2 . The method of  claim 1 , further comprising the step of
 (vi) discarding the urine, saliva or mouthwash that has passed through the filter.   
     
     
         3 . The method of  claim 1 or 2 , wherein the urine sample is whole urine, preferably wherein the whole urine is micturated 1 hour or less before filtering the urine in step (iii). 
     
     
         4 . The method of  any one of the previous claims , wherein the sample preparation system comprises a pre-filter and wherein the sample passes through the pre-filter before passing through the filter. 
     
     
         5 . The method of  any one of the previous claims , wherein amplifying DNA or RNA in the lysate may comprise amplifying DNA or RNA of one or more bacteria, fungi, protists, or viruses, preferably one or more bacteria or protists causing STDs or urinary tract infections. 
     
     
         6 . The method of  any one of the previous claims , wherein amplifying DNA in the lysate comprises extracting the lysate from the filter before amplification, preferably wherein the sample preparation unit comprises one or more outlets through which the lysate may be transferred from the filter to an amplification unit in which the DNA amplification is performed. 
     
     
         7 . The method of  claim 6 , further including the step of establishing a liquid connection between the one or more outlets of the sample preparation unit and an inlet of the amplification unit. 
     
     
         8 . The method of  any one of the previous claims , wherein the filter comprises a first side and a second side, wherein filtering the sample through the filter comprises forcing the sample from the first side to the second side by applying a pressure on the sample at the first side that is above atmospheric pressure, preferably wherein the pressure is applied by a mechanical means, more preferably wherein the mechanical means involves moving a piston within a chamber of the sample preparation system. 
     
     
         9 . The method of  any one of the previous claims , wherein step (iii) comprises passing at least 5 ml, at least 10 ml, at least 20 ml, or at least 50 ml of the sample through the filter. 
     
     
         10 . The method of  any one of the previous claims , wherein step (iv) further comprises applying a pressure above atmospheric pressure to the lysate buffer when applying it to the filter, preferably wherein the pressure is provided by mechanical actuation of a piston or pouch. 
     
     
         11 . The method of  any one of the previous claims , wherein the filter has a pore size no greater than 1.0 μm, preferably no greater than 0.8 μm, more preferably no greater than 0.6 μm, even more preferably no greater than 0.5 μm, most preferably no greater than 0.45 μm; alternatively the filter has a pore size no greater than 0.4 μm, and more preferably no greater than 0.22 μm. 
     
     
         12 . The method of  any one of the previous claims , wherein step (iv) further comprises applying a washing buffer to the filter, preferably wherein the washing buffer comprises purified water. 
     
     
         13 . The method of  any one of the previous claims , wherein the lysis buffer comprises at least one of sodium hydroxide, sodium dihydrogen phosphate, disodium hydrogen phosphate, Tris, HEPES, and detergent, more preferably wherein the lysis buffer comprises at least 250 mM NaOH. 
     
     
         14 . The method of  any one of the previous claims , wherein step (iv) further comprises applying a neutralization reagent to the filter, wherein the neutralization reagent comprises a composition for neutralization of the pH value of the sample, preferably wherein the neutralization reagent comprises an alkaline reagent and/or wherein the neutralization buffer comprises one or more of Tris, Tris-HCl, and ethylenediaminetetraacetic acid (EDTA). 
     
     
         15 . The method of  any one of the previous claims , wherein step (iv) further comprises applying a rehydration buffer to the filter, preferably wherein the rehydration buffer comprises purified water, more preferably wherein the rehydration buffer comprises water having one or more of KCl, ammonium sulfate, MgSO4, and deoxynucleotide triphosphates, detergent, Triton X-100, and betaine. 
     
     
         16 . The method of  any one of the previous claims , wherein step (v) comprises amplifying DNA by Loop-Mediated Isothermal Amplification (LAMP).